Deacetylasperulosidic Acid Ameliorates Pruritus, Immune Imbalance, and Skin Barrier Dysfunction in 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis NC/Nga Mice

The prevalence of atopic dermatitis (AD), a disease characterized by severe pruritus, immune imbalance, and skin barrier dysfunction, is rapidly increasing worldwide. Deacetylasperulosidic acid (DAA) has anti-atopic activity in the three main cell types associated with AD: keratinocytes, mast cells, and eosinophils. Our study investigated the anti-atopic activity of DAA in 2,4-dinitrochlorobenzene-induced NC/Nga mice. DAA alleviated the symptoms of AD, including infiltration of inflammatory cells (mast cells and eosinophils), epidermal thickness, ear thickness, and scratching behavior. Furthermore, DAA reduced serum IgE, histamine, and IgG1/IgG2a ratio and modulated the levels of AD-related cytokines and chemokines, namely interleukin (IL)-1β, IL-4, IL-6, IL-9, IL-10, IL-12, tumor necrosis factor-α, interferon-γ, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated on activation the normal T cell expressed and secreted in the serum. DAA restored immune balance by regulating gene expression and secretion of Th1-, Th2-, Th9-, Th17-, and Th22-mediated inflammatory factors in the dorsal skin and splenocytes and restored skin barrier function by increasing the expression of the pro-filaggrin gene and barrier-related proteins filaggrin, involucrin, and loricrin. These results suggest DAA as a potential therapeutic agent that can alleviate the symptoms of AD by reducing pruritus, modulating immune imbalance, and restoring skin barrier function.

In patients with AD, the expression and secretion of the AD-related cytokines interleukin (IL)-25, IL-33, thymic stromal lymphopoietin (TSLP), and the chemokines thymus and activation-regulated chemokine (TARC), macrophage-derived chemokines (MDC) and, regulated on activation, normal T cell expressed and secreted (RANTES) are increased in keratinocytes [10,11]. The cytokines IL-25, IL-33, and TSLP can stimulate Th2 cells directly Figure 1A shows representative images of the six test groups, including the normal, control, predinisolone (PD), and 3, 6, and 12 mg/kg DAA groups during weeks 10-14 after induction of AD. Repetitive application of DNCB aggravated the clinical symptoms associated with AD, including erythema, maceration, dryness, abrasions, lichenification, scratching behavior, and ear thickness, over the first nine weeks. The dermatitis score, scratching behavior, and ear thickness were significantly higher in the control group than in the normal group. DAA administration significantly reduced these parameters in a dose-dependent manner ( Figure 1B-D), resulting in similar efficacy in the 3 mg/kg DAA and PD groups on week 14. The clinical features of AD in DNCBinduced NC/Nga mice, that is, (B) dermatitis score, (C) scratching behavior, and (D) ear thickness were evaluated thrice a week during administration of carboxymethyl cellulose (CMC), prednisolone (PD), and DAA. The results were expressed as the mean ± standard deviation (n = 6). ### p < 0.001 vs. normal (naïve control group); ** p < 0.01 and *** p < 0.001 vs. control (negative control; DNCB-treated group), PD (positive control; DNCB + prednisolone 3 mg/kg), and DAA (DNCB + DAA 3, 6, or 12 mg/kg) treatment groups; and † p < 0.05, † † p < 0.01, and † † † p < 0.001 vs. PD treatment group.

DAA Reduces the Thickening of the Epidermis, along with Eosinophil and Mast Cell Infiltration
Epidermal thickness and the number of infiltrated inflammatory cells were investigated to confirm the efficacy of DAA in the dorsal skin of NC/Nga mice. Epidermal thickness and the number of infiltrated inflammatory cells were significantly higher in the control group than in the normal group. DAA treatment reduced the epidermal thickness and the number of infiltrated inflammatory cells in a dose-dependent manner, resulting in similar efficacy between the 3 mg/kg DAA and PD groups ( Figure 2). The graph shows the epidermal thickness of each group. (C) The graph was displayed by measuring the number of infiltrated inflammatory cells (mast cells and eosinophils) in each group. Histological analysis and the number of inflammatory cells were examined in six randomized dorsal skin sites from each mouse. The results are expressed as the mean ± standard deviation (n = 6). ### p < 0.001 vs. normal (naïve control group); *** p < 0.001 vs. control (negative control; DNCB-treated group), PD (positive control; DNCB + prednisolone 3 mg/kg), and DAA (DNCB + DAA 3, 6, or 12 mg/kg) treatment groups; and † p < 0.05 and † † p < 0.01 vs. PD treatment group.

DAA Reduces Immunoglobulin E (IgE) and Histamine Levels in the Serum of NC/Nga Mice
IgE and histamine levels were significantly higher in the control group treated with DNCB than in the normal group. DAA decreased serum IgE and histamine levels in a dose-dependent manner. DAA exhibited a higher inhibitory effect on IgE and histamine than PD (Figure 3). Effects of DAA on serum IgE and histamine levels. Serum IgE and histamine levels were measured using enzyme-linked immunosorbent assay (ELISA). The results were expressed as the mean ± standard deviation (n = 6). ### p < 0.001 vs. normal (naïve control group); *** p < 0.001 vs. control (negative control; DNCB-treated group), PD (positive control; DNCB + prednisolone 3 mg/kg), and DAA (DNCB + DAA 3, 6, or 12 mg/kg) treatment groups; and † † p < 0.01 and † † † p < 0.001 vs. PD treatment group.

DAA Restores the IgG1/IgG2a Balance in the Serum
The restoration of Th1/Th2 balance was investigated by measuring the levels of serum IgG1 and IgG2a on DAA treatment. The serum IgG1 level of the DNCB-induced control group was significantly higher than that of the normal group. DAA reduced serum IgG1 levels in a dose-dependent manner, with similar efficacy between the 3 mg/kg DAA and PD groups ( Figure 4A). Furthermore, the serum IgG2a level of DNCB-induced mice was significantly higher than that of the normal mice. When compared to the control group, the DAA group displayed a dose-dependent increase in IgG2a levels, whereas IgG2a levels decreased in the PD group ( Figure 4B). DAA reduced IgG1 and increased IgG2a levels; therefore, the level of IgG1/IgG2a was normalized in a dose-dependent manner. In the PD group, both IgG1 and IgG2a levels were reduced, resulting in a decrease in the IgG1/IgG2a ratio ( Figure 4A-C).

DAA Regulates Serum Levels of AD-Related Cytokines and Chemokines
The levels of the cytokines IL-4, IL-9, IL-10, and TSLP secreted by activated Th2 cells, and the levels of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) involved in acute and chronic AD were significantly higher in the control group than in the normal group. Furthermore, the levels of the chemokines TARC, MDC, and RANTES, which directly or indirectly activate Th2 cells, were significantly increased in the control group compared to levels in the normal group. DAA decreased the levels of cytokines and chemokines in a dose-dependent manner, resulting in a similar efficacy between the 3 mg/kg DAA and PD groups ( Figure 5A-C). Th1-mediated cytokines IL-12 and IFN-γ were significantly decreased in the control group compared to levels in the normal group. DAA restored serum IL-12 and IFN-γ levels in a dose-dependent manner compared to levels in the control group. We found that 12 mg/kg DAA restored the level of IL-12 and IFN-γ to that observed in the normal group ( Figure 5D). DAA reduced Th2 and Th9-related cytokines and improved AD by increasing Th1-mediated cytokines. However, PD reduced Th1-, Th2-, and Th9-mediated cytokines and chemokines.

DAA Regulates the Secretion Levels of AD-Related Cytokines in Splenocytes
The levels of Th2 cytokines IL-4, IL-5, IL-10, IL-13, IL-31, IL-33, and TSLP were significantly higher in the control group than in the normal group. Moreover, the levels of acute and chronic AD-related IL-1β, IL-6, TNF-α, TARC, MDC, and RANTES were significantly higher in the control group than in the normal group. Furthermore, the levels of Th9-, Th17-, and Th22-related cytokines IL-9, IL-17, and IL-22 were significantly higher in the control group than in the normal group. DAA reduced the levels of ADrelated cytokines and chemokines in a dose-dependent manner, resulting in a similar efficacy between the 3 mg/kg DAA and PD groups ( Figure 6A-D). Th1-mediated cytokines IL-12 and IFN-γ were significantly decreased in the control group compared to levels in the normal group. DAA restored the secretion levels of IL-12 and IFN-γ in a dosedependent manner compared to levels in the control group. We found that 12 mg/kg DAA restored the secretion level of IL-12 and IFN-γ to that observed in the normal group ( Figure 6E). DAA modulated the balance of AD-related cytokine and chemokine production in splenocytes, while PD decreased Th1, Th2, Th9, Th17, and Th22-mediated cytokine and chemokine production.

DAA Regulates the Gene Expression of AD-Related Cytokines and Chemokines in the Dorsal Skin of NC/Nga Mice
The gene expression of Th2-mediated cytokines IL-4, IL-5, IL-10, IL-13, and IL-31 was significantly higher in the control group than in the normal group. Gene expression of the acute and chronic AD-related IL-1β, IL-6, and TNF-α as well as Th9-, Th17-, Th22-related IL-9, IL-17, and IL-22, respectively, was significantly increased in the control group compared to levels in the normal group. DAA treatment reduced the gene expression of AD-related cytokines and chemokines, including Th2-mediated cytokines, in a dose-dependent manner, resulting in a similar efficacy between the 3 mg/kg DAA and the PD groups ( Figure 8A-C). Th1-mediated cytokines IL-12 and IFN-γ were significantly decreased in the control group compared to levels in the normal group. DAA restored the gene expression levels of IL-12 and IFN-γ in a dose-dependent manner compared to levels in the control group. We found that 12 mg/kg DAA restored the gene expression level of IL-12 and IFN-γ to that observed in the normal group ( Figure 8D). DAA treatment attenuated immune imbalance by modulating AD-related cytokine and chemokine gene expression in the dorsal skin, while PD treatment decreased AD-related cytokine and chemokine gene expression. The results were expressed as the mean ± standard deviation (n = 6). ## p < 0.01, and ### p < 0.001 vs. normal (naïve control group); * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (negative control; DNCB-treated group), PD (positive control; DNCB + prednisolone 3 mg/kg), and DAA (DNCB + DAA 3, 6, or 12 mg/kg) treatment groups; and † p < 0.05, † † p < 0.01, and † † † p < 0.001 vs. PD treatment group.

DAA Alleviates the Defects of the Skin Barrier Function
The expression of skin barrier-related FLG, IVL, and LOR proteins was significantly lower in the DNCB-induced control group than in the normal group. DAA treatment increased the abundance of proteins in a dose-dependent manner compared to that in the control group ( Figure 9A,C). The gene expression of pro-FLG was significantly decreased in the DNCB-induced control group compared to that in the normal group. DAA treatment increased pro-FLG gene expression in a dose-dependent manner ( Figure 9B). We found that 12 mg/kg DAA restored the expression of skin barrier-related proteins and pro-FLG gene to a level similar to that in the normal group. However, PD slightly restored the expression of the skin barrier proteins FLG, IVL, and LOR when compared to DAA ( Figure 9A,C). The results are expressed as the mean ± standard deviation (n = 6). ### p < 0.001 vs. normal (naïve control group), * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control (negative control; DNCB-treated group), PD (positive control; DNCB + prednisolone 3 mg/kg), and DAA (DNCB + DAA 3, 6, or 12 mg/kg) treatment groups, † p < 0.05, † † p < 0.01 and † † † p < 0.001 vs. PD treatment group, and @@@ p < 0.001 vs. normal (naïve control group). FLG, filaggrin; IVL, involucrin; LOR, loricrin; pro-FLG, pro-filaggrin.

Discussion
In the present study, the efficacy of DAA against AD was investigated using an in vivo animal model, with a particular focus on the mechanism by which DAA alleviates ADrelated clinical symptoms, scratching behavior, immune imbalance, and recuperation of skin barrier function. Pruritus is an important clinical feature of AD and is one of the main diagnostic criteria. Controlling the "itch-scratch" cycle is important in the treatment of AD and is associated with a reduction in clinical symptoms, immune cell infiltration, ear thickness, and epidermal thickening [46]. DAA treatment successfully improved ADrelated clinical symptoms, ear thickness, and scratching behavior (Figure 1). Moreover, histological analysis showed a decrease in epidermal thickness and the number of infiltrated inflammatory cells (Figure 2). These results suggest that DAA may be clinically useful in AD treatment. Furthermore, serum IgE and histamine levels decreased after DAA treatment, indicating that DAA was effective in reducing pruritus by reducing the levels of IgE and histamine, which are involved in itching (Figure 3).
Antigen penetration through the defective epidermal barrier activates keratinocytes, mast cells, and eosinophils, which secrete cytokines, including IL-4 and IL-13, to increase Th2 cell activity [47][48][49][50]. DAA treatment reduced the gene expression and secretion of Th2 cytokines, including IL-4, IL-5, IL-13, and IL-31, in the dorsal skin and splenocytes, thereby reducing Th2-mediated cytokine levels in serum ( Figures 5A, 6A and 8A). The decrease in IL-4 and IL-13 levels was consistent with the decrease in serum IgG1 levels ( Figure 4A). Th1-mediated cytokines IL-12 and IFN-γ are associated with the activity of Th1 cells [51]. IL-12 activates Th1 cells, and IFN-γ secreted from Th1 cells reduces Th2 cell activity and IL-4 secretion, thereby suppressing IgE and IgG1 production and inducing IgG2a production in B cells [52,53]. DAA treatment increased the levels of the Th1-mediated cytokines IL-12 and IFN-γ in the serum, splenocytes, and dorsal skin ( Figures 5D, 6E and 8D). Thus, DAA increased the level of serum IgG2a by decreasing IL-4 and IL-13 and increasing Th1mediated cytokines ( Figure 4B). Consequently, DAA treatment normalized the IgG1/IgG2a ratio in a dose-dependent manner ( Figure 4C). In the PD group, the IgG1/IgG2a ratio was restored by the reduction of IgG1 and IgG2a. Collectively, our results suggest that PD restored the immune balance by inhibiting both Th1 and Th2 cell activity, whereas DAA restored the immune balance by regulating Th1 and Th2 cell activity. Reduction of the Th2-mediated cytokines IL-5 and IL-31 suggests that DAA reduced eosinophil infiltration, differentiation, and survival, and inhibited the progression of chronic AD. Furthermore, we suggest that DAA may alleviate clinical symptoms, particularly pruritus, through the reduction of IL-31.
The AD-related cytokines, namely IL-1β, IL-6, and TNF-α, are expressed and secreted by inflammatory cells such as keratinocytes, Th2 cells, mast cells, and eosinophils. They are involved in the progression of acute and chronic AD and promote the activation of Th9 [54], Th17, and Th22 cells [55][56][57]. DAA treatment reduced the gene expression and secretion levels of IL-1β, IL-6, and TNF-α in the serum, splenocytes, and dorsal skin ( Figures 5B, 6B and 8B). These results demonstrate that DAA alleviated AD by reducing cytokine levels and inhibiting the activities of Th9, Th17, and Th22 cells involved in acute and chronic AD.
Th9-mediated IL-9 promotes the expression and secretion of IL-4 in several inflammatory cells, such as keratinocytes and mast cells [26,28]. Furthermore, IL-9 promotes mast cell and eosinophil infiltration [26,29,58]. In this study, DAA treatment reduced the levels of IL-9 in the serum, splenocytes, and dorsal skin ( Figures 5A, 6C and 8C), indicating that DAA inhibited the activation of Th9 cells, thereby reducing the cytokine IL-4 associated with Th2 cells. This is consistent with the decrease in the number of infiltrating mast cells and eosinophils (Figure 2). Th2-mediated cytokines IL-4 and IL-10 promote the production of IL-9 in Th9 cells [27]. Our study suggests that DAA reduced both the gene expression and secretion levels of IL-4 and IL-10, thereby suppressing Th9 cell activity. Th1-mediated IFN-γ levels have been reported to be inversely related to IL-9 levels [59]. Our study showed a decrease in IFN-γ levels and an increase in IL-9 levels in the control group, while DAA treatment decreased IL-9 levels and increased IFN-γ levels ( Figures 5, 6 and 8). These results suggest that DAA restores immune balance by decreasing IL-9 levels and increasing IFN-γ levels.
IL-17 and IL-22 are mainly related to the activation of Th2 cells and impair skin barrier function [32]. DAA treatment reduced the secretion of IL-17 and IL-22 from splenocytes and dorsal skin ( Figures 6C and 8C), suggesting that DAA restores immune balance by inhibiting the activity of Th2 cells and suppressing the activity of Th17 and Th22 cells. In addition, DAA restored the expression of skin barrier-related proteins in the dorsal skin ( Figure 9).
Keratinocytes induce a biased Th2 immune response, which further exacerbates immune imbalance and pruritus in patients with AD [60]. TARC, MDC, and RANTES are secreted by keratinocytes, mast cells, and eosinophils, and may be involved in the chronicization of AD [11,16]. DAA reduced the expression of TARC, MDC, RANTES, and CCR4 (receptor for TARC and MDC) in serum, splenocytes, and dorsal skin ( Figures 5C, 6D and 7). This indicates that DAA can reduce the binding of TARC and MDC by reducing CCR4 expression. Recently, IL-17 and IL-22 have been reported to increase CCR4 expression [32,61,62]. Our results demonstrated that a reduction in IL-17 and IL-22 levels decreased CCR4 expression. Collectively, the reduction of TARC, MDC, and CCR4 suppresses the activity of Th2 cells, indicating that it is effective in restoring immune balance. Reduction of RANTES reduced the growth and differentiation of eosinophils, thereby alleviating the symptoms of chronic AD and epidermal thickening [16,63,64].
IL-33 and TSLP secreted primarily from keratinocytes are increased in AD and stimulate Th2 cells, mast cells, and eosinophils [65,66]. IL-25 is mainly secreted by keratinocytes and promotes the production of Th2-mediated cytokines IL-4 and IL-5 in mast cells [67]. DAA reduced the gene expression and secretion of these cytokines in the serum, splenocytes, and dorsal skin ( Figures 5A, 6A and 7B). These results correlate with the decrease in infiltrated mast cells and eosinophils (Figure 2). Cytokines and chemokines produced by several immune cells, including Th2, Th9, Th17, and Th22, reduce the expression of the skin barrier-related proteins FLG, IVL, and LOR [5,6,26,34,68,69]. DAA increased the gene expression of pro-FLG and the expression of skin barrier-related proteins. In contrast, PD treatment resulted in a slight increase in skin barrier-related proteins compared to that in the control group (Figure 9). Previous studies have reported that TCS increases the skin barrier related proteins FLG and LOR [70]. PD is thought to restore skin barrier-related proteins through the reduction of AD-related cytokine levels. DAA showed a higher efficacy than PD treatment for both gene expression of pro-FLG and restoration of skin barrier-related proteins. Unlike PD, DAA treatment might alleviate AD by restoring the beneficial immune balance and skin barrier function. Overall, we suggest that DAA may be a potential anti-AD drug candidate with higher efficacy and lower toxicity than PD.

Animals
Four-week-old male NC/Nga mice were purchased from Shizuoka Laboratory Center Inc. (Shizuoka, Japan). Mice were maintained on a 12 h light/dark cycle at 25 ± 5 • C and 50% ± 5% humidity in individually ventilated cages in a specific pathogen-free facility at Central Laboratory Animal and had ad libitum access to food and water (Catalog number 5L79, Central Laboratory Animal, Seoul, Korea). Serum collection was performed according to methods described in previous studies [6,7]. Briefly, on the last day of the experiment, mice were anesthetized with isoflurane (2-2.5%), and blood was immediately collected from the venous vessels, followed by euthanization using CO 2 . Blood was stored at room temperature for 1 h and centrifuged at 3000× g at 4 • C for 15 min to supernatant of serum. Serum samples were stored at −80 • C until use. All experimental procedures were performed according to the protocol approved by the Kyung Hee University Animal Care and Use Committee guidelines (approval number KHSASP-21-088, date of approval: 23 February 2021).

Application of AD-Like Skin Lesions and Treatment of DAA
DNCB application and DAA treatment were performed through the methods of previous studies [6,7]. Briefly, after acclimatization for 1 week, hair was removed from the dorsal skin of NC/Nga mice using an electric razor. Mice were randomly divided into normal (DNCB untreated group; naïve control), control (DNCB-treated group; negative control), PD (prednisolone; positive control), DAA 3, 6, and 12 mg/kg groups, and six mice were assigned to each group. To induce AD-like skin lesions, 1% DNCB was dissolved in a mixture of acetone and ethanol (2:3) and applied twice to the dorsal skin (200 µL) and right ear (100 µL) of the mice. After sensitization, 0.4% DNCB was dissolved in a mixture of acetone and olive oil (3:1) and applied repeatedly to the skin on the back (150 µL) and right ear (50 µL) thrice a week for 14 weeks. At week 10, mice in the normal and control groups were orally administered 0.5% carboxymethyl cellulose (CMC) daily. Additionally, 3 mg/kg PD and 3, 6, or 12 mg/kg DAA were each dissolved in CMC and orally administered daily for four weeks. The schedule for DNCB induction and oral administration of DAA is shown in Figure 10.

Dermatitis Score and Ear Thickness
Dermatitis scores were recorded three times per week based on previous studies [71]. Clinical symptoms were evaluated with a score of 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of the five symptoms (clinical symptoms: erythema, maceration, dryness, abrasions, and lichenification). The total dermatitis score was quantified as the average of all individual scores for the five symptoms. Ear thickness was measured thrice a week in the right ear of each mouse using an ear thickness gauge (Mitutoyo Corporation, Tokyo, Japan). Figure 10. Schematic diagram of DNCB treatment and oral administration of DAA. Four-weekold male NC/Nga mice were acclimated for 1 week, and the hair on the dorsal skin was shaved. Thereafter, 1% DNCB was applied twice to the dorsal skin and right ear. Thereafter, 0.4% DNCB was applied thrice a week until the 14th week. DAA and PD were orally administered from weeks 10-14.

Scratching Behavior
The scratching behavior of NC/Nga mice was recorded three times per week [72]. Briefly, the mice were acclimatized to acrylic cages for 1 h after vehicle administration. Scratching behavior with the hind paw was then measured and recorded on the skin on the neck, ears, and back for 30 min. Scores ranging from 0-4 (i.e., 0 points (none), 2 points (less than 1.5 s), 4 points (1.5 s or more) were assigned. The total score for scratching behavior was determined as the average of the individual measures.

Histological Analysis
Histological analysis was performed according to previously described methods [6,7,68]. Briefly, mice were sacrificed, and the dorsal skin was fixed with 10% formalin. Tissues were sectioned to a thickness of 4 µm and stained with hematoxylin and eosin and toluidine blue. After staining, the sections were observed under an optical microscope (400×, DP Controller Software, Olympus Optical, Tokyo, Japan). The epidermis thickness and the number of infiltrated inflammatory cells (e.g., mast cells and eosinophils) were measured at six sites per mouse using Image J software (National Institute of Health, Starkville, MD, USA).

Serum Immunoglobulin and Histamine Assay
The levels of IgE, histamine, IgG1, and IgG2a in the serum were measured using a mouse enzyme-linked immunosorbent assay (ELISA) kit. The levels of all cytokines and chemokines were measured using a mouse ELISA kit according to the manufacturer's instructions.

Splenocyte Supernatant Isolation and Cytokine and Chemokine Analysis
Splenocytes were crushed and examined as described previously [6,7,68]. Briefly, splenocytes were disrupted with the back of a sterile syringe plunger and harvested using a cell strainer. Splenocytes were treated with red blood cell lysis buffer. Thereafter, the splenocytes were washed three times with RPMI-1640 medium supplemented with 10% FBS. The isolated splenocytes were treated with 5 µg/mL Con-A, seeded in 24-well plates at a concentration of 1 × 10 6 cells/well, and incubated for 72 h at 37 • C with 5% CO 2 . Supernatants were collected, and splenocytes were homogenized in lysis buffer containing cOmplete™ protease inhibitor cocktail tablets. The lysate was centrifuged at 4 • C and 10,000× g for 10 min. After centrifugation, the splenocyte supernatant and lysate were frozen at −80 • C for subsequent cytokine and chemokine analyses. Cytokines IL-1β, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17, IL-22, IL-31, IL-33, TSLP, TNF-α, IFN-γ, TARC, MDC, and RANTES present in the supernatant were measured using an ELISA kit, according to the manufacturer's instructions. The lysate protein concentration was measured using the Pierce™ BCA protein assay kit. The levels of cytokines and chemokines in the supernatants were normalized to the protein concentration of the lysate.

RNA Extraction and Quantitative Real Time-Polymerase Chain Reaction (RT-qPCR)
RNA extraction and cDNA synthesis were performed as described previously [7]. Briefly, total RNA was extracted from the dorsal skin of mice using the Easy-Red total RNA extraction kit. Chloroform was added to the extract and stored at room temperature (25 ± 5 • C) for 20 min. The supernatant was collected by centrifugation for 15 min at 10,000× g at 4 • C, and an equal volume of isopropanol was added to the supernatant and stored overnight. After 24 h, centrifugation was performed for 15 min at 10,000× g at 4 • C and washed with 75% ethanol. After removing the ethanol, the extract was dried at room temperature (25 ± 5 • C) for 1 h and then dissolved in diethyl pyrocarbonate water. Complementary DNA was synthesized using a cDNA synthesis kit and RT-qPCR was performed on an ABI StepOnePlus™ real-time PCR system (Applied Biosystems, Waltham, MA, USA) using the synthesized cDNA and SYBR Premix EX Taq. The primer sequences are listed in Table 1. The mRNA expression level was normalized to that of GAPDH using the 2 −∆∆Ct method to obtain the cycle threshold (Ct) value.

Protein Extraction and Western Blotting
The dorsal skin was isolated, and examinations were performed as previously described [6,7]. Briefly, the dorsal skin tissues frozen in liquid nitrogen were crushed using a pestle. Subsequently, the skin tissues were homogenized in lysis buffer containing cOm-plete™ protease inhibitor cocktail tablets. The lysates of dorsal skin tissues were sonicated and centrifuged at 10,000× g for 15 min at 4 • C. The concentration of protein in the supernatant was quantified using the Pierce™ BCA protein assay kit. After quantitation, equal amounts of protein were loaded on 12% SDS-PAGE for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 and incubated overnight at 4 • C with primary antibodies (FLG, LOR, IVL, and β-actin) at a dilution of 1:1000. The following day, the membranes were treated with an HRP secondary antibody for 2 h at a dilution of 1:5000 and visualized using a ChemiDoc™XRS + System (Bio-Rad, Richmond, CA, USA). The expression level of each protein was analyzed using Image Lab statistical software (Bio-Rad, CA, USA) and normalized to the expression of β-actin.

Statistical Analysis
Data are presented as the means ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's honestly significant difference test. Statistically significant differences were evaluated using SPSS (SPSS Inc., Chicago, IL, USA).